The principal objective of the research project is to achieve molecular understandings of biological energy transduction, particularly the synthesis of ATP by oxidative and photosynthetic phosphorylation. The main thrust of the experiments is to test the concept that ATP is formed at the catalytic site by reversal of hydrolysis, coupled to energy-linked protein conformational changes. Approaches used will include millisecond mixing and quenching and pulse-chase experiments to determine if inorganic phosphate and ATP may be transitorily but tightly-bound to the catalytic site, during steady-state phosphorylation. These and other experiments will explore a postulated alternating dual site mechanism in which a tightly-bound ATP is released at one site concomitant with a change in Pi binding at a second site in a manner that favors ATP formation. A new series of experiments will be directed toward elucidating the mechanism of the O18 exchange, particularly to test the working hypothesis that it results from the reversible cleavage of bound ATP. Use will be made of ATP labeled in the beta-gamma bridge oxygen, with search for mixing of this oxygen with other beta-phosphoryl oxygens that should accompany a reversible cleavage of the bound ATP. In addition, measurements will be made with highly-labeled inorganic phosphate with mass determination of the complete phosphate molecule to determine the number of oxygen atoms lost from each phosphate during an exchange cycle. Other experiments will test the effect of uncouplers on the various exchange reactions with chromatophores and chloroplasts to find if observations made with mitochondria can be generalized. In a related system, the O18 exchange of pyrophosphatase will be probed to find if indication of formation of bound metaphosphate in the presence of manganese can be substantiated. Efforts will be continued to obtain crystalline preparations of the chloroplast ATPase suitable for cooperative x-ray studies. BIBLIOGRAPHIC REFERENCES: Boyer, P.D., "Energy Transduction and Proton Translocation by Adenosine Triphosphatases," FEBS Letters 50, 91-94 (1975). Boyer, P.D., "A Model for Conformational Coupling of Membrane Potential and Proton Translocation to ATP Synthesis and to Active Transport," FEBS Letters 58, 1-6, (1975).